Affinity binding of a vampire bat plasminogen activator to SEC resins.
DSPAalpha1 is a recombinant form of the vampire bat plasminogen activator which we have produced in mammalian cell culture. During the development of a recovery process for DSPAalpha1 we observed an unexpected binding interaction between this protein and several types of gel filtration chromatography resins. Under typical operating conditions using neutral pH buffers, we found that DSPA flows through the sizing resin and is fractionated, as expected, according to its molecular size. However, DSPA applied under certain acidic conditions (<pH 3) binds tightly to the Sephacryl series of resins. The protein is not released until solvent conditions are changed, specifically the pH is raised above 3. From the results presented we conclude that this unexpected interaction with the gel filtration media is not simply an ion exchange nor a hydrophobic interaction, but rather a more complex, mixed mode "affinity" like binding. Several structural features of the DSPA protein which may be involved in this unique binding have been examined, including binding after inactivation of its active site, chemical deglycosylation, chemical denaturation, or limited proteolysis. The "affinity" interactions persist despite these treatments and lead us to conclude that there may be a unique peptide sequence(s) within the protein which is responsible for the binding interaction to Sephacryl resins.
McCaman
M T
MT
Process Development Department, Berlex Biosciences, 15049 San Pablo Avenue, Richmond, California 94804-00990, USA. mike_mccaman@berlex.com
Souders
C
C
Ottoboni
S
S
Pungor
E
E
D000180:Acrylic Resins; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002685:Chiroptera; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D010960:Plasminogen Activators / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D000180:Acrylic Resins; D011994:Recombinant Proteins; C037239:Sephacryl Superfine; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D010960:Plasminogen Activators